Design of Peptides with High Affinities for Heparin and Endothelial Cell Proteoglycans

Abstract

Proteoglycan-binding peptides were designed based on consensus sequences in heparin-binding proteins: XBBXBX and XBBBXXBX, where X and B are hydropathic and basic residues, respectively. Initial peptide constructs included (AKKARA) n and (ARKKAAKA) n (n ‫؍‬ 1-6). Affinity coelectrophoresis revealed that low M r peptides (600 -1300) had no affinities for low M r heparin, but higher M r peptides (2000 -3500) exhibited significant affinities (K d Х 50 -150 nM), which increased with peptide M r . Affinity was strongest when sequence arrays were contiguous and alanines and arginines occupied hydropathic and basic positions, but inclusion of prolines was disruptive. A peptide including a single consensus sequence of the serglycin proteoglycan core protein bound heparin strongly (K d Х 200 nM), likely owing to dimerization through cysteine-cysteine linkages. Circular dichroism showed that high affinity heparin-binding peptides converted from a charged coil to an ␣-helix upon heparin addition, whereas weak heparin-binding peptides did not. Higher M r peptides exhibited high affinities for total endothelial cell proteoglycans (K d Х 300 nM), and ϳ4-fold weaker affinities for their free glycosaminoglycan chains. Thus, peptides including concatamers of heparin-binding consensus sequences may exhibit strong affinities for heparin and proteoglycans. Such peptides may be applicable in promoting cell-substratum adhesion or in the design of drugs targeted to proteoglycan-containing cell surfaces and extracellular matrices. Proteoglycans (PGs)1 are composed of a core protein to which are covalently attached one or more sulfated glycosaminoglycans (GAGs). PGs are ubiquitous components of cell surfaces and the extracellular matrix, and their GAG chains contribute to myriad biological functions, such as modulation of enzyme activities, regulation of cell growth, and control of assembly of the extracellular matrix (1). PGs are thus potential targets for therapeutic intervention. For example, heparin antagonists are needed to take the place of protamine, a heterogeneous, sometimes toxic protein mixture commonly used to neutralize the anticoagulant activity of heparin in humans (2, 3); in the design of drugs to be targeted to PG-rich tissues, such as cartilage (4); and to be used to promote cell adhesion in a variety of situations, e.g. by promoting binding of cells that express abundant amounts of PGs, such as endothelial cells (5), to synthetic vascular graft surfaces. To develop a rationale for the design of such reagents, it is useful to examine known features of protein structure required for high affinity interactions with GAGs. Thus, analysis of the structural features of many known heparin-and heparan sulfate (HS)-binding proteins has revealed the presence of conserved motifs, through which GAG binding has been postulated to occur. Cardin and Weintraub (6) identified two clusters of basic charge in known heparin-binding proteins in which amino acids tend to be arranged in the patterns XBBXBX or XBBBXXBX, where B represents an ...

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